The sporulation and germination-outgrowth developmental cycle of the bacterium Bacillus subtilis provides a model biological system for examining intracellular differentiation. This project is focused on two specific aspects of this developmental cycle. (1) Control or transfer RNA synthesis and regulatory roles of tRNA. Specific approaches include an examination of the chromosomal organization of the tRNA genes, as examined by restriction enzyme methodology and gene cloning, and an analysis of isoacceptor tRNA synthesis using the techniques of two dimensional gel electrophoresis and RNA-DNA hybridization. (2) Factors regulating gene expression during spore development. A mechanism which either alters the capacity of DNA to serve as a template for RNA transcription, or one which controls RNA polymerase itself may be involved in the ordered expression of enzyme observed during spore outgrowth and spore formation. Specific approaches to further examine this question will include the cloning of the structural gene sequences and promoter elements for several enzymes which have specific points of synthesis during the developmental cycle. An examination of the transcriptional specificity of RNA polymerase for these sequences will be made.